A comparison of three spermatozoa selection techniques for Intracytoplasmic Sperm Injection (ICSI) using swim-up, Cumulus Oophorus Model and PICSI ® Dish

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Intracytoplasmic Sperm Injection

(ICSI) using Swim-up, Cumulus

Oophorus Model and PICSI ® Dish.

by

Ms Michelle Rijsdijk

Dissertation presented for the degree of Doctor in Reproductive Medical Science in the

Faculty of Health Science,

Department of Obstetrics and Gynaecology at Stellenbosch University

Supervisor:

Prof DR Franken

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Declaration

The development and writing of the papers (published and unpublished) were the principal responsibility of myself and, for each of the cases where this is not the case, a declaration is included in the dissertation indicating the nature and extent of the

contributions of co-authors.

Signature:………..

Date:……December 2015………..

By submitting this dissertation electronically, I declare that the entirety of the work

contained therein is my own, original work, that I am the sole author thereof (save

to the extent explicitly otherwise stated), that reproduction and publication thereof

by Stellenbosch University will not infringe any third party rights and that I have

not previously in its entirety or in part submitted it for obtaining any qualification.

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Abstract

Introduction: Spermatozoa selection for Intracytoplasmic Sperm Injection (ICSI) is a

paramount factor in the outcome of a fertility treatment cycle. Nature has perfected the selection process by using the cumulus matrix to select spermatozoa that are morphologically and genetically normal.

Aim: To determine which method of semen preparation delivers the best results in terms of

spermatozoa selection for ICSI.

Methods: Patients were randomized into 3 groups of spermatozoa selection techniques namely

the routine swim-up or density gradient, the Cumulus Model or the PICSI® dish (hyaluronic acid). The prepared collected spermatozoa were used to make slides to record the percentage of normal spermatozoa (morphological staining), the capacitational status (chlorotetracycline test), chromatin packaging quality (chromomycin A3 (CMA3)staining) and the DNA quality

(acridine orange staining). These results were then compared to the fertilization, cleavage, pregnancy and implantation rates of the patients used in the study.

Results: All three groups displayed improvements in morphology, capacitational ability,

chromatin packaging quality and DNA quality (or fragmentation). There was no significant difference in pregnancy rates between the groups and no difference in implantation rates. The PICSI® group did however show a significant improvement in the chromatin packaging quality, only if the baseline values were low.

Conclusions: All 3 groups of spermatozoa selection techniques showed improvements in

spermatozoa quality. The swim-up/gradient group showed a statistical improvement in the fertilization rate when compared to the cumulus and PICSI® groups. PICSI® showed a greater improvement in selected spermatozoa parameters when baseline values for CMA3

were low.

Keywords: ICSI, cumulus oophorus, PICSI®_{, morphology, capacitation, acrosome}

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Opsomming

Inleiding: Die seleksie van spermatosoa vir Intrasitoplasmatiese Sperm Inspuiting (ICSI) is

'n deurslaggewende faktor in die uitkoms van 'n fertiliteitsbehandeling-siklus. Die natuurlike seleksieproses van morfologies en geneties normale sperme, word bepaal deur die cumulus oophorus matriks.

Doel: Om die beste metode van semen voorbereiding te bepaal, waardeur optimale resultate

in terme van seleksie van spermatosoë kan plaasvind vir ICSI.

Metodes: Pasiënte is ewekansig in drie groepe van spermseleksie-tegnieke verdeel, naamlik

die opswemtegniek, die Cumulus Model of die PICSI® Dish (Hialuroon suur). Die

voorbereide spermatosoa is gebruik om mikroskoopskyfies te maak vir die bepaling van die persentasie, normale morfologiese spermatosoë, die kapasitasie status (Chlorotetrasikline toets), chromatien verpakking kwalitiet (Chromomycin A3 kleuring) en die DNA-integriteit (Akridien Oranje kleuring). Die resultate is vergelyk tov die bevrugting-, verdeling-, swangerskap- en inplantasie syfers van die pasiënte in die studie.

Resultate: Al drie groepe het verbeterings getoon ten opsigte van morfologie, kapasitasie,

chromatien verpakking kwaliteit en DNA fragmentasie. Daar was geen beduidende verskil in swangerskap of inplantasie syfers tussen die groepe nie. Die PICSI® groep het 'n statisties betekenisvolle verbetering getoon as die CMA3 waarde laag was voor die seleksie metode.

Gevolgtrekkings: Al drie spermatosoë seleksie tegnieke het verbeterings getoon in

spermatozoa kwaliteit. Die opswemtegniek-groep het 'n statistiese betekenisvolle verbetering in die bevrugtingsyfer getoon in vergelyking met die PICSI en cumulus metodes. Die PICSI® tegniek het ‘n betekenisvolle verbetering getoon as waardes vir CMA3 laag.

Sleutelwoorde: ICSI, cumulus oophorus, PICSI®_{, morfologie, kapasitasie,}

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Acknowledgements

I wish to extend my most sincere gratitude and appreciation to the following people for their contribution to the successful completion of this study:

Professor Danie Franken, for the fountain of knowledge that you made available to me, and

the jokes that kept me smiling,

Professor TF Kruger, for your support, guidance and sheer determination to help, words

will never be able to aptly say Thank You

Dr Carl Lombard of the MRC, for calculating the statistical outcomes of the study,

Drs Paul Dalmeyer and Danie Botha, and the patients of Fembryo Fertility Clinic for the

financial support and unwavering sense of encouragement,

To Coopers Africa, for sponsoring the purchase of some of the PICSI® dishes,

To Life Healthcare St Georges, for sponsoring the purchase of some of the PICSI® dishes, To Lloyd Goddard of Vivid Air, for purchasing chemicals for the staining procedures, To the Staff of Fembryo Fertility Clinic, for all your support over the years,

To James Wilde, for designing a selection of my illustrations,

To my co-worker and colleague, Wilhelm Schoeman, for always being the helping hand and always letting me see the other side of the coin,

To my sister, Maryke McDonald, for all the typing and checking and re-checking,

To my family, Rudy and Nell Braam, as well as my sister, Maryke, and brother-in-law,

Sean McDonald, for all your love and support over the years……. We have finally done it!!!

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List of Figures and Tables

Figure 1: A representation of spermatogenesis 10

Figure 2: A representation of the mechanisms following ejaculation from

capacitation to the acrosome reaction 13

Figure 3: A diagram depicting the difference between the cytoplasmic retention of a

diminished mature spermatozoa and that of a normal spermatozoa 16

Figure 4: A diagram depicting the maturation of an ooctye and its polar bodies 20

Figure 5: The hatching blastocyst on Day 5 of development 30

Figure 6: A photo of the ICSI process whereby one spermatozoon is injected into a metaphase

II oocyte. 34

Figure 7: Embryo culture from Day 0 to Day 5 35

Figure 8: An illustration of the swim-up and gradient methods of spermatozoa preparation 40

Figure 9: Experimental design of the cumulus oophorus model 42

Figure 10: Microdot of PICSI dish where the spermatozoa are adhering to the hyaluronic

acid (HA) droplet. This patient shows good adherence. 43

Figure 11: Illustration of the use of the PICSI® dish. 44

Figure 12: A longitudinal section and schematic drawing of a normal spermatozoon. 46

Figure 13: A photograph of the CMA3 stain illustrating the difference between

good (dull stain) and poor (bright stain) quality spermatozoa in terms of

DNA packaging quality 48

Figure 14: The staining pattern CTC 5, showing the acrosome reacted spermatozoa 50

Figure 15: A photo of the staining characteristics of the acridine orange stain (AO) 51

Figure 16: An illustration of the study design 52

Figure 17: An example of the randomization chart used to assign patients to the study 53

Figure 18: The statistically significant measure of change from pre- to post-preparation for

morphology (p < 0.05) 56

Figure 19: The measure of change from pre- and post-preparation for CTC in all groups 57

Figure 20: The measure of change from pre- and post-preparation for CMA3 in all groups. 58

Figure 21: The measure of change from pre- and post-preparation for AO in all three groups 60

Figure 22: The fertilization rate in the three groups with the average = solid line. 61

Figure 23: The pregnancy rate as distributed across the three groups per transfer 63

Figure 24: The decline in pregnancy rate as seen with increasing age in all three groups 64

Table 1: The results of the tests performed for each of the study groups 54

Table 2: The differences for each group for all the tests done pre- and post-preparation 55

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Chapter 1: Literature Review

Since the introduction of intracytoplasmic sperm injection (ICSI) (Palermo et al. 1992) to the assisted reproductive technology (ART) arena, couples exhibiting a severe male factor have been able to accomplish their reproductive goal. ICSI has managed to bypass a number of key factors relating to the fertilization process concerning the spermatozoa. Unfortunately, the pregnancy rate is not optimal in terms of professional expectations, thus prompting continued investigation into improvement of success rates. The recruitment of follicles and ovarian stimulation cannot be altered to improve embryo quality at the stage of laboratory handling; spermatozoa selection is the major factor that can be manipulated. This study looked at the selection of spermatozoa for ICSI using three different selection techniques. All have been extensively researched but never compared to each other in terms of quality, namely, acrosome reaction (AR), morphology, DNA packaging quality and DNA quality, and fertilization, pregnancy and implantation rates.

The aim of this study was two-fold:

Primary aim: - to determine which of the three relatively cost-effective techniques

delivered the best results in terms of spermatozoa quality.

Secondary aim: To compare the fertilization, embryo quality and pregnancy rates of the

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1.1. The Spermatozoon

Spermatogenesis in the adult male requires many processes resulting in primary spermatocytes after a series of cellular differentiations (see Figure 1). After the first meiotic division, the primary spermatocytes will form two secondary spermatocytes, dividing for a second time into secondary spermatocytes which are haploid in nature (Youssef et al. 1996). Spermiogenesis (maturation of the spermatids) then follows the spermatogenesis, and both processes are completed in approximately 74 days (Acosta et al. 1990a; Acosta et al. 1990b). Not all spermatogonia are destined to become

spermatocytes, as some fail to complete the entire process.

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Spermatozoon chromatin is incredibly stable and compact. To achieve this condensed format, the spermatozoa DNA is specifically organized to allow transfer of the densely packaged genetic material to the oocyte. This format of packaging is due to significant modifications of the nucleoprotein compartments and replacement of histones by protamines (Ward & Coffey, 1991; Bouvier, 1977; Balhourn, 1989). Protamines are arginine-rich proteins that primarily neutralize the DNA charge and compact the

chromatin (Mali et al. 1988) and this is complemented by the formation of intra- and inter-molecular disulphide cross-links between the cysteine residues. Once the spermatozoa reaches the epididymis, protamine synthesis occurs and all protamines have been dephosphorylated (Claasens, 1991).

The mature spermatozoon morphologically consists of a head with acrosome, the mid-piece and the tail regions (Figure 1). These regions play important roles in normal

fertilization and their criteria have been extensively explained according to the Tygerberg Strict Criteria (Kruger et al. 1986; Kruger et al. 1996; Menkveld et al. 1990).

The head is oval shaped with a length of 4 – 5.5μm and width of 2.5 – 3.5μm (Menkveld et al. 1990). Numerous variations are considered normal but anomalies such as large heads, small heads (pinheads), tapering, amorphous and vacuolated heads (Menkveld & Coetzee, 1995) have also been identified. The nucleus constitutes 65% of the head where the DNA is linked with other proteins to form a chromatin string so that the chromosomes are no longer visible (Kruger et al. 1996; Menkveld & Kruger, 1996; Weinbauer et al. 2000). The acrosomal region covers the anterior two-thirds of the head (arising originally from the golgi complex) (Claasens, 1991; Kruger et al. 1986; Kruger et al. 1996;

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The mid-piece contains mitochondria providing the energy needed for the spermatozoa motility and is approximately the same length as the head, separating the head and tail regions (Kruger et al. 1996; Weinbauer et al. 2000).

The tail, or flagellum, has 9 evenly spaced, peripheral, doubled microtubules and a central pair of single microtubules. The tail is the longest region with a length of 4 – 10μm and a diameter of 1μm (Kruger et al. 1996)

Human spermatozoa, unlike those of other species, have to undergo a series of physiological and morphological changes once ejaculated, called capacitation, before fertilization can take place (Loeb, 1915; Yanagimachi, 1994). These changes are

mandatory along with hyperactivation (a unique, vigorous motility) of the spermatozoa, to enter the oocyte and its vestments (Yanagimachi, 1988; Yanagimachi, 1994).

Hyperactivation is a pattern of movement seen in the spermatozoa which is a distinctive, vigorous motility as first described by Yanagimachi in 1969 (Yanagimachi et al; 1969). This movement involves increased flagellar bend amplitude and beat symmetry.

Capacitation is imperative to prevent spermatozoa from becoming fertile too quickly, given that during coitus, spermatozoa are deposited in the vagina and the distance the spermatozoa covers, to get to the oocyte, is extensive. Once capacitation has occurred, it allows for commencement of the AR (DasGupta et al. 1993).

Capacitation is a series of the following processes:

1. removal of the spermatozoa surface proteins (as seen by the green bars surrounding the spermatozoa in Figure 2), allowing better for better binding of the spermatozoa to the oocyte

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2. efflux of cholesterol from the spermatozoon membrane, allowing for improved permeability of Ca2+

3. changes in the oxidative metabolism

4. hyperactivity (more whip-like movement of the tail)

5. increases in phosphotyrosine phosphorylation of certain proteins 6. decreases in calmodulin binding proteins

7. increases in Ca2+_{uptake (using the Ca}2+_{-ATPase pumps)}

8. increases in intracellular pH and lastly increases in cyclic adenosine monophosphate (cAMP) levels (DasGupta et al. 1993; Overstreet, 1996).

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Multiple steroids help to induce the Ca2+-influx with one of them being identified by Osman et al. (1989), as 4-pregnen-3,20-dione (progesterone) and 4-pregnen-17alpha-ol-3,20-dione (17-alpha-hydroxyprogesterone) which are present at the fertilization site in

vivo. The AR is an enzymatic activity resulting in increases in intracellular Ca2+ allowing

acrosomal exocytosis and therefore leading to successful fertilization (DasGupta et al. 1993).

There are 5 stages of the AR:

Stage 1: Intact acrosome: Acrosomal membranes are intact and the acrosomal matrix is

homogenous.

Stage2: Decondensed matrix: sealing of the matrix while the outer acrosomal

membranes remain intact.

Stage 3: Vesicles and matrix: Vesicles are present within the acrosomal cap and

rearrangement of the plasma and outer acrosomal membranes take place (as seen by the last spermatozoa in Figure 2)

Stage 4: Fusion: The outer acrosomal and plasma membranes fuse to each other in the

cap region of the equatorial segment. Disappearance of the matrix is also evident.

Stage 5: Inner acrosomal membrane exposed: The plasma and outer acrosomal membranes

have vanished; the equatorial segment is still usually intact.(Claassens, 1991; Nagae et al. 1986).

Capacitation and AR are therefore the two fundamental steps that the spermatozoon needs to complete before fertilization can occur. Cumulus cells have been shown to stimulate the AR (Hong et al. 2009) as spermatozoa that have been co-incubated with cumulus have lower numbers with intact acrosomes.

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1.1.1.

The Molecular Selection of Mature Spermatozoa using Hyaluronic Acid

Spermatozoa have been known to suffer from physiological and environmental stressors which lead to morphological aberrations, gene mutations and chromosomal abnormalities (Evenson et al. 2002) which ultimately lead to a disruption in the biochemical events leading up to and including fertilization as well as embryogenesis. Spermatozoa

chromatin abnormalities induce chromatin structural problems and could lead to apoptosis and necrosis (Darzynkiewicz et al. 1997) which arise during spermiogenesis if the DNA nicking and ligating activities are abnormal (Evenson et al. 2002).

Other biochemical processes and thus possible markers that can be used to determine the maturity of the spermatozoon have been carefully researched. Creatine kinase (CK) is one of the markers that has been found to be higher in men with lower fertility potential

(oligozoospermic males) (Huszar et al. 1988). Autoradiographic and CK immunostaining (Cayli et al. 2003; Huszar and Vigue, 1993) have provided an explanation that increased CK and other protein concentrations suggest that a developmental defect has occurred in the terminal stages of spermatogenesis. Increases in CK, as well as other cytoplasmic protein concentrates, cause process malfunctions in the extrusion of the cytoplasm (see Figure 2); ‘Residual bodies’, otherwise known as cytoplasmic droplets, are left in the adluminal area. These cytoplasmic droplets are therefore a visual indication of immaturity of the spermatozoon. Huszar et al. (1992) showed in their in vitro fertilization (IVF) study that all normozoospermic men with high levels of CK did not achieve successful pregnancy results vs. other patients in the normozoospermic group. This was in agreement with their earlier study (Huszar and Vigue; 1990) which looked at the correlation of intrauterine insemination (IUI) pregnancy rates with spermatozoa

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parameters; they found that the spermatozoa CK activity was a significant predictor of outcome in comparison to other parameters such as count and motility.

Figure 3: A diagram depicting the difference between the cytoplasmic retention of a diminished mature spermatozoon and that of a normal spermatozoon.

Through subsequent research of Huszar and Vigue (1990), another protein was

identified as the 70KDa testis-expressed heat shock protein known as HspA2 (Huszar et al. 2000). This heat shock protein, HspA2, first appears in primary and secondary spermatocytes, and concurrently with the expression of HspA2, major protein movements occur and result in cytoplasmic extrusion and remodelling of the

spermatozoa plasma membrane (Huszar et al. 1997; Huszar et al. 2000, Huszar et al. 2007). This supports the theory that HspA2 supports chromosomal crossing-over as it is responsible for the delivery of DNA repair enzymes as well as histone-protamine

replacement in terms of the membrane remodelling (Ergur et al. 2002; Jakab et al.

HspA2 expression Plasma membrane remodeling

No HspA2

Cytoplasmic Retention

Cytoplasmic Extrusion

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2005). Lack of this testis-specific chaperone (HspA2) can lead to meiotic errors (Nixon et al. 2015). Anomalies in the expression of HspA2 have been seen to be concurrent with an increase in numerical chromosomal aberration (Sakkas et al. 1991 (1); Sakkas et al. 1999 (2)), higher levels of reactive oxygen species (ROS) (Huszar et al. 1993) and increases in DNA chain fragmentation (Aitken et al. 1994; Nixon et al. 2015). These have all been supported by the discovery that low levels of HspA2 expression are more prevalently seen in men suffering severe oligo-astheno-teratozoospermia (OATS) (Griffin et al. 2003; Jakab et al. 2005, Twigg et al. 1998)

The HspA2 also acts as a hyaluronan receptor during normal fertilization (Huszar et al. 2003; Parmegiani et al. 2008). It has been shown that hyaluronan-bound spermatozoa (spermatozoa bound to the PICSI dish) have increased developmental maturity,

increased chromatin integrity, increased morphology, increased functional competence (Keel et al. 1987), decreased aneuploidy, and decreased active caspase-3 (Cayli et al. 2003; Jakab et al. 2005; Worrilow et al. 2009; Worrilow et al. 2013). The Petersen group from Brazil (2008 and 2010) do not agree with these findings and have published several articles discrediting the efficacy of the PICSI dish.

This protein, localized in several organs, is found on the spermatozoa’s surface (Ghosh et al. 2003). With the phosphorylation of hyaluronan-binding protein -1 (HABP-1) there is a reversal in the production of D-mannosylated albumin (which prevents the spermatozoa-egg binding) and this action therefore promotes sperm-egg binding (a type of cell signalling) (Ghosh et al. 2007). HABP-1 is a 34kDa glycosaminoglycan

consisting of 209 amino acids (Ghosh et al. 2007) and is produced in the seminiferous tubules as a pro-protein (Bharadwij et al. 2002). The protein is located in many bodily organs and but more specifically on the spermatozoa surface and also acts as a

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mannose-binding site for zona pellucida (ZP) recognition (Ghosh et al. 2003; Ghosh et al. 2007). Samples that have <20% motility (asthenozoospermia) have been shown to be deficient in HABP-1 and therefore there is a significant decrease in the spermatozoa fertilization potential (Ghosh et al. 2003).Conversely, the oocyte’s granulosa cells also express HABP-1 and are seen to increase during ovulation; this results in the expansion of the cumulus oophorus complex (COC) which in turn exerts an effect on the follicular fluid (Thakar et al. 2006).

Apoptosis is another important regulator of normal spermatogenesis; it can be explained as programmed cell death or a controlled disassembly of cells from within (Seli et al. 2004). Apoptosis starts with movement of negatively charged phospholipids of the plasma membrane from the inner surface to the outer surface (Seli et al. 2004). This is associated with the activation of caspases and the cytosolic cysteine-containing

aspartate-specific proteases (De Fried & Danaday, 2010). Fas, a cell-surface protein, then binds to FasL, which is produced by the Sertoli cells, and results in apoptosis of the spermatozoon (Seli et al. 2004). Apoptosis then leads to the breaking of the double-stranded DNA within the spermatozoon and the process of self-demise is complete (Ahmadi & Ng, 1999).

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1.2 The Oocyte and its Vestments

Mature human oocytes are in a constant state of meiotic arrest (at prophase) (Mortimer& Swan, 1995; Overstreet 1996; Veeck 1986). This continues until puberty when the ovaries are activated by hormones.

These hormones, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), are produced by the anterior pituitary gland. Only oogonia that are ovulated will complete the first meiotic division resulting in two daughter cells, each with haploid number of chromosomes. The first is known as the oocyte as it retains most of the ooplasm and the second, the first polar body (Mortimer, 1995; Veeck, 1986). The second division takes place after ovulation of the Graafian follicle, only if the oocyte is fertilized (Fatchi et al. 2002; Nagae et al. 1986; Veeck, 1986) and the second polar body will then become visible.

The maturation characteristics of the metaphase II oocyte are as follows (Figure 4) 1. Extrusion of the first polar body

2. Symmetrical ooplasm which is homogenous in colour and smooth granularity 3. The cumulus matrix should appear as a ‘halo’ around the oocyte with an expanded

coronal layer

4. Expansion of the cumulus mass

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Figure 4: A diagram depicting the maturation on an oocyte and its polar bodies Picture by J Wilde

The maturation processes are not just determined by the nucleus, but also by its vestments (Hong et al. 2004; Sousa et al. 1997) and this is evident by the hormonal impact that oestrogen (changes the reactivity of Ca2+ release systems) and progesterone have on the maturity of the cumulus matrix as well as the ooplasm (Overstreet, 1996; Sousa et al. 1997). Cumulus cells have been shown to play an imperative role in oocyte maturity, specifically with regard to the nuclear and cytoplasmic maturity (Tesarik et al. 1997) and it has been shown that oestrogen (E2) exertion results in Ca2+ oscillations during

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The cumulus oophorus complex (COC) is made of two components, the inner stratum, made of trypsin-sensitive granules embedded in a viscoelastic matrix comprised of hyaluronic acid (HA) (Cooper & Yeung, 2000; Fatchi et al. 2002) and the outer stratum, consisting of the glycoproteins called the zona pellucida (ZP). The importance of the cumulus matrix is mainly responsible for secreting cAMP which is key in maintaining meiosis after the second division and protecting the oocyte from environmental effects (Overstreet, 1996). The cAMP acts via gap junctions and these mechanisms are cut off when the cumulus cells retract from the ZP near the time of ovulation (Menkveld & Coetzee, 1995; Overstreet, 1996; Fatchi et al. 2002). The COC is also known to facilitate oocyte transport in the fallopian tubes, as well as improving the spermatozoon’s fertilizing ability (Yanagimachi 1994) by among others activating capacitation and thus leading to the AR of the spermatozoon both through hormonal (oestrogen and progesterone) and mechanical mechanisms (Foresta et al. 1992).

This is theorized to aid in the selection of morphologically normal spermatozoa (Fatchi et al. 2002). Research has corroborated this (Carrell et al. 2000; Fetterolf et al. 1994; Mansour et al. 1995; Stock et al. 1989; White et al. 1990); spermatozoa that have been retrieved from the cumulus matrix were partially acrosome reacted (Cooper & Yeung, 1995; Hong et al. 2004; Rijsdijk & Franken, 2007) and the presence of cumulus cells has resulted in selection of better spermatozoa (Carrell et al. 2000; Corselli & Talbot, 1987; Stock et al. 1989; Suarez et al. 1983). This selection of morphologically superior spermatozoa can still not be fully explained as the matrix has many components, namely, hormonal/steroidal impact, mechanical and dimensional. The chemotactic/chemokinetic factors (hormonal/steroidal) can be demonstrated in follicular fluid (Ralt et al. 1994; Cohen-Dayag et al. 1994) but this does not explain the effect of the cumulus and more importantly the matrix as a whole on the selection of the spermatozoa.

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The extracellular matrix of the COC is formed primarily of hyaluronan (a dermantan sulphate proteoglycan), but also contains other proteins such as inter-alpha-trypsin inhibitor, adermatan sulphate proteoglycan, and a pentraxin-3. (Drahorad et al. 1991; Relucenti et al. 2005). The matrix is comprised of the hyaluronan and the proteins and thus aids in gamete interactions (Relucenti et al. 2005).

The effect of HA has been extensively researched and has been shown to:

1. improve developmental maturity of spermatozoa, increase the quantity of acrosome-reacted spermatozoa (by increasing intracellular Ca2+),

2. improve spermatozoa chromatin integrity, 3. increase spermatozoa morphology,

4. increase functional competence of spermatozoa (by stimulating spermatozoa motility),

5. decrease chromosomal aneuploidy and decrease active caspase-3 of spermatozoa (Bains et al. 2001; Cayli et al. 2003; Jakab et al. 2005)

Carell (2000) showed that zygotes derived from ICSI oocytes showed better embryo development when co-cultured with cumulus cells. It has also been shown in mice that in the presence of a hyaluronidase inhibitor, cumulus cell dispersal is inhibited (Kim et al. 2005); this possibly influences oocyte maturity.

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1.3 Fertilization In Vivo

Fertilization in vivo incorporates several processes in a specific order of events including spermatozoa penetration through the cumulus matrix, spermatozoon binding to the ZP, acrosomal exocytosis, spermatozoa penetration through the ZP and fusion of the

spermatozoa and oocyte (Yanagimachi et al. 1994). Capacitation of the spermatozoa is required, with the acrosome still intact, to facilitate the spermatozoa to transverse the cumulus matrix. The matrix therefore acts as a natural ‘selector’ of spermatozoa by allowing only a selected spermatozoon populace through to the ZP. The exact

mechanisms and the extent to which the cumulus acts are still not clear. As seen in IVF, the natural selection process of spermatozoa leads to fertilization (Benchaib et al. 2003; Jakab et al. 2005) unlike that of ICSI.

The cumulus then stimulates the beginning of the AR (this takes place at the ZP) as well as increased motility. This increased motility is needed for the spermatozoa to travel through the cumulus as well as the ZP. The extracellular matrix of the cumulus mass is high in HA and it is believed that this acid facilitates the spermatozoa’s passage through the matrix. Binding of the acrosome-intact spermatozoa to the ZP induces the fusion of the plasma and outer acrosomal membranes, also known as the acrosome reaction (AR). This process allows for interaction with the ZP and allows entry into the oocyte (Kim et al. 2005). The process of spermatozoa incorporation into the oolemma is phagocytotic in nature and only acrosome-reacted spermatozoa have the capability to fuse with the oolemma. A cascade of subsequent events leads to a rise in intracellular Ca2+ levels, which in turn results in the exocytosis of cortical granules from the ooplasm, which

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causes chemical changes in the ZP, which in turn make the ZP impenetrable to other spermatozoa. This fusion has therefore led to the ‘activation’ of the oocyte.

A few hours later, the pronuclei of both the gametes can be visualized and this is called the pronuclear stage (Veeck & Zaninovic 2003). The zygotic centrosome is then assembled, a vital process that leads to the final progression of the fertilization procedure whereby there is reorganization and union of the maternal and paternal chromosomes and formation of the zygote (Veeck et al. 2003). The human zygote remains single-celled for 24 hours and the fertilization process is completed when the first cleavage occurs.

In vitro fertilization leads to a ‘natural’ selection process of spermatozoa, leading to fertilization (Benchaib et al. 2003; Jakab et al. 2005). The fertilization process may require as little as one hour of spermatozoa exposure to the oocyte (Gianaroli et al. 1996) in comparison to ICSI.

1.4 Intracytoplasmic Sperm Injection (ICSI)

1.4.1 Spermatozoa Selection for ICSI

ICSI manages to bypass many processes during fertilization compared to in vivo fertilization.

The exceptional fertilization rate of ICSI is offset by the occasional lack of blastocyst development if damaged spermatozoa have been injected into the oocytes. Damaged spermatozoa have been shown to cause adverse events such as: fertilization failure, early

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embryo death, spontaneous abortion, and childhood cancers, as well as infertility in resulting offspring due to chromosomal aneuploidies and propagation disorder linked to

Y-chromosome deletions (Jakab et al. 2005). This can be explained by the natural selective nature of the cumulus matrix during IVF (Benchaib et al. 2003; Jakab et al. 2005) as ICSI shows a fivefold increase in chromosomal aberrations, 8% increase in spontaneous abortions (IVF has abortion rate of 10% and ICSI 18%) and increases in malformation of embryos (Jakab et al. 2005).

It has been postulated that spermatozoon DNA damage (Muratori et al. 2000; Sakkas et al. 2003) as the result of aborted apoptosis, although this has been challenged. Sakkas et al. (2003) suggested that spermatozoa with DNA fragmentation, ‘escaped’ apoptosis. Along the same lines, Marcon and Boissoneault (2004) proposed that DNA damage was due to the incorrect reparation of transient DNA nicks that occur during

spermatogenesis. Many other factors have been shown to cause DNA fragmentation including advanced paternal age, certain drugs (e.g. chemotherapy), smoking, genital tract inflammation and varicoceles (Sakkas and Alvarez, 2010). DNA fragmentation influences fertilization rates for the threshold value above 10% as well as implantation rates (Benchaib et al. 2003). The DNA fragmentation index (DFI) is a predictive factor for ART outcome (Bungnum et al. 2007). DFI below 30% showed no increased risk in early pregnancy loss but, when the DFI was more than 30%, ICSI was indicated as the preferred method of treatment (Bungnum et al. 2007; Parmegiani et al. 2010a and Parmegiani et al. 2010b; Zini et al. 2008). Benchaib et al. (2003) compared ICSI to IVF and stated that DNA fragmentation only plays a role in the embryo development of ICSI embryos when DNA fragmentation is severe, where Tomsu et al. (2002) was in

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They stated that ICSI patients receive ICSI as preferred treatment because of their decreased parameters (such as motility, forward progression and morphology) and therefore displayed increased spermatozoa defragmentation rates (Benchaib et al. 2003; Gandini et al. 2000; Irvine et al. 2000; Sun et al. 1997;. Gandini et al. (2004) however suggested that the stringent spermatozoa selection process in ICSI as well as the embryo selection mitigated the negative effects of the DNA damage. This was evident in the research of Hirsch et al. (1999) who stated that ICSI patients with high rates of DNA fragmentation had longer time to pregnancy (TTP) than other ICSI patients; this was confirmed by the meta-analysis of Zini et al. (2009) that showed that both ICSI and IVF patients with high DFI had an increased rate of pregnancy loss.

There have been several attempts to try to improve ICSI results, especially when spermatozoa parameters are exceptionally low. These research studies have resulted in several novel techniques. One such technique, as in this study, is using the PICSI® dish for spermatozoa selection. The PICSI® dish has been shown to select spermatozoa with better DNA chromatin packing as described by Huszar et al. (Huszar et al. 1997; Huszar et al. 2000, Huszar et al. 2007). The mechanisms are explained on from page 15.

Another technique, intracytoplasmic morphologically selected sperm injection (IMSI) (Bartoov et al. 2002), with the emphasis on spermatozoa morphology (at ultra-high magnifications), has seen a significant improvement in pregnancy rates. Contrasting views and results have been published relating to the results of IMSI. Khattabi et al. (2013) reported improved pregnancy rates but this was only seen when poor

spermatozoa parameters led to embryo degradation after day 3 of development, indicating the impact of the ‘late’ paternal effect (Bartoov et al. 2002; Khattabi et al.

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2013). In contrast Oliveira et al. (2011) could not show an improvement in fertilization, embryo quality, pregnancy and implantation although a statistical trend was noted toward improved ongoing pregnancies, live births and lower miscarriage rates. The equipment required for IMSI is however costly, and laboratories with limited budgets will not be able to implement this new technique very easily.

In comparison, another technique in use is the selection of ZP-bound spermatozoa for ICSI (Liu et al. 2011). This Chinese and Australian collaboration (Liu et al. 2011) reported higher numbers of good quality embryos and higher implantation rates. The spermatozoa were allowed to bind to immature oocytes and after manipulation the bound spermatozoa were used for ICSI. The results obtained were as theoretically expected, as spermatozoa that bind to the ZP have been shown to have normal morphology as well as normal DNA quality (Menkveld et al. 1991). This study has limitations as the study group was small (a total of 53 couples per group).

Gianaroli et al. (2008) used yet another technique: ‘sperm head birefringence’ (SHBF) to select spermatozoa for ICSI. Spermatozoa have anisotrophic properties and the mature spermatozoa’s nucleus can be determined using double refraction of light to view the pattern of human SHBF and DNA damage (Gianaroli et al. 2008; Petersen et al. 2010). Gianaroli et al. (2008) studied several different groups including

oligozoospermic patients with no progressive motility, as well as patients undergoing testis biopsy. This study (Gianaroli et al., 2008) showed a higher number of grade 1 embryos on day 3 (higher rate of good quality embryos) as well as a higher implantation rate than the control group in all the different spermatozoa quality samples. The

pregnancy rate for the control group was initially higher; however the abortion rate was also increased, thus illustrating that abnormal genetic complement leads to spontaneous

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abortion. A later study (Gianaroli et al. 2010) showed that there was an increase in the implantation rate of the cycles where the reacted spermatozoa were being used. This is a method that has merit because it can be used across the male diagnosis spectrum. The equipment required for this method is however expensive and not suited to laboratories with limited budgets.

Said et al. (2005a, 2005b, 2006 and 2008) highlighted the use of another non-invasive selection technique called magnetic activated cell sorting (MACS). This cell sorting uses annexin V-conjugated microbeads to remove spermatozoa that are apoptotic based on the externalization phosphatidylserine residues. During one specific apoptotic event, phosphatidylserine, which is normally present in the inner leaflet of the sperm-plasma membrane, is externalized, and this binds to the annexin V beads therefore ‘marking’ the spermatozoon as apoptotic (Paasch et al. 2003). A case study by Rawe et al. (2010) showed a pregnancy result for a patient with high levels of spermatozoon DNA

fragmentation when the MACS spermatozoa selection method was used. More recently Troya et al. (2015) showed that using spermatozoa with intact membranes and a normal nucleas (after MACS selection) for ICSI delivered a higher pregnancy rate (58.1%) compared to PICSI (40.4%) as well as to normal ICSI (27.3%) (Troya et al. 2015)

1.4.2 Fertilization In Vitro

During ICSI, spermatozoa are selected by the embryologist and injected into the ooplasm. Previously it was thought that the oocyte supplied all the primary materials, in the form of proteins and mRNA, and that spermatozoa played a secondary role. The activated maternal genome (by the spermatozoon) is in force during the fertilization of the oocyte and the first cleavage of the embryo, the paternal genes are ‘switched on’ at the 4-cell stage until the

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blastocyst stage, when the embryonic genome is activated (Ahmadi & Ng, 1999; Seli et al. 2004).

Lopes et al. (1998) showed that with DNA damage of more than 25%, the fertilization rate is less than 20% (normal fertilization is seen to be between 60 and 98%). Oocytes do however have a limited capacity to repair DNA damage (originating from the spermatozoa) but this is related to the extent of the spermatozoa damage (Ahmadi & Ng, 1999), as well as the

maternal age. Elevated levels of spermatozoa DNA damage or advanced maternal age have been shown to result in increased embryonic fragmentation (Sakkas et al. 1996) or arrest. The presence of vacuoles in the spermatozoon head have also been shown to negatively affect fertilization rates as reported by Sakkas et al. (1998) and Lopes et al. (1998), who were investigating the selection of spermatozoa using IMSI. The vacuoles have been thought to be correlated to spermatozoa DNA packaging as well as quality. Conflicting reports have been published in recent years, showing increases in fertilization rates with no effect on pregnancy, and others showing improvements in pregnancy rates, but not in embryo development when PICSI® was used for spermatozoa selection (Nasr-Esfahani et al. 2008; Parmegiani et al. 2010; Tarozzi et al. 2009; Ye et al. 2006). This discrepancy may be due to different study designs, data analysis and patient populations (Said & Land, 2011). Oocytes have been shown to have the capacity to repair spermatozoa DNA damage (Ahmadi &Ng, 1999; Genesca et al. 1992) but this depends on the extent of the damage as well as the age and competence of the oocyte (Sakkas et al. 1996).

1.4.3 Cleavage

Cleavage of cells is a universal feature which occurs after nuclear replication and segregation (Veeck et al. 2003). The human prezygote undergoes mitotic divisions

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every 12-18 hours (Veeck et al. 2003) and this process is controlled by the spermatozoa centrosome. The cytoplasm characteristically elongates and the exterior membrane contracts around the smallest circumference with the narrowing continuing until the zygote is divided into two separate blastomeres (Veeck et al. 2003).

Bartoov et al. (2002) showed that the spermatozoa nucleus is the most important factor affecting ART outcome. It is very rare for cleavage not to take place once pronuclear formation has been noted, but this event occurs in less than 5% of normally fertilized oocytes. This has been attributed to centrosomal problems of the spermatozoon. This is evident when oocytes inseminated using ICSI have failed to progress past the pronuclear stage, but when the same oocytes are inseminated using IVF, they cleave normally. This is due to insufficient spermatozoa aster formation (Chemes & Sedo, 2011). Because the spermatozoa head-neck junction cannot be visualized by the operator performing the ICSI, the spermatozoa selection is less than optimal.

1.4.4 Blastocyst Formation

Blastocyst formation starts between 114 and 120 hours after injection of the spermatozoon into the oocyte. The hatching of a human blastocyst takes place at approximately 144 hours (Veeck et al. 2003) and can be seen in Figure 5. Blastocyst development was originally thought to be controlled only by the embryonic genome, but it was recently made evident that blastocycst formation genetically also falls into the realm of the ‘late paternal’ effect of the spermatozoon (Seli et al. 2004). The rate of blastocyst development varies significantly from 20.9% to 52.3% (Rubio et al. 2014). Shoukir et al. (1998) reported that blastocyst rates decrease in ICSI patients versus patients undergoing IVF and this was confirmed in subsequent studies by Sakkas et al. (1998), Miller & Smith (2001). Embryos of good quality (the grading on day 2 and 3)

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that subsequently degenerate reflect the effects of abnormalities in the paternal genome (Jones et al. 1998). Vanderzwalmen et al. (2008) observed that blastocyst rates

increased with the use of IMSI. This was explained by the decrease in spermatozoa DNA fragmentation (and thus the late paternal effect) resulting in better blastocyst formation rates (Jones et al. 1998).

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Chapter 2: Materials and Methods

2.1 Patients and Study Groups

2.1.2 Inclusion and Exclusion Criteria

All couples (n = 223) who were included in the study underwent ICSI as the method of treatment (teratozoospermic patients). The patients were randomly divided into three groups (73 in the swim-up group; 73 in the cumulus group and 77 in the PICSI group).

The group allocation was as follows:

1. ICSI using the conventional double wash swim-up method (Group 1) 2. ICSI using the cumulus oophorus capillary model (Group 2)

(As described by Hong et al. 2004 and later modified by Rijsdijk and Franken, 2007) 3. ICSI using the hyaluronan droplets for selection (PICSI® dish) (Group 3)

Once the patient had been allocated a spermatozoa selection technique using the randomization table (as seen on page 53), slides were made of the raw (Before) and prepared samples (After). The slides were then treated appropriately for each of the tests as laid out in Section 2.3. All patients in the study signed an informed consent form after Institutional Review Board approval. Patients were only granted access to their results once the study was completed.

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2.1.3 Ovulation Induction

Female patients had a wide variety of diagnosis including anovulation and

endometriosis. Female patients were not subject to any exclusion criteria as we wanted to report research results based on a general IVF arena. Ovarian stimulation in an agonist cycle (long protocols) was carried out by the administration on day 18 (of previous cycle) of gonadotrophin-releasing hormone (GnRHa) (Lucrin Depot®_{; Abbott}

Laboratories South Africa (Pty) Ltd), followed by 14-15 days of pure

follicle-stimulating hormone (FSH, 75 IU) and luteinizing hormone (LH, 75 IU) (Menopur®_,

Ferring Pharmaceuticals, South Africa, (Pty) Ltd) from day 2 of the cycle. Patients were followed up by doing a series of ultrasonographical measurements of the Graafian follicles. Ovulation was then induced by the administration of human chorionic

gonadotrophin (hCG) (10 000 IU) (Ovidrel®; Serono, South Africa (Pty) Ltd) when the leading Graafian follicles reached 18-20mm. The oocytes were then aspirated 36 hours after administration of the hCG.

Stimulation in the antagonist cycles (short protocols) was carried out by the administration of pure FSH (75 IU) and LH (75 IU) (Menopur®, Ferring

Pharmaceuticals, South Africa, Pty. Ltd.) from day 2 of the cycle. LH suppression was achieved using Cetrotide 3mg (Merck (Pty) Ltd) on day 8 of the cycle and Cetrotide 0.25mg (Merck (Pty) Ltd) on day 11. The antagonist cycle then proceeded to aspiration with administration of hCG (Ovidrel®; Serono, South Africa (Pty) Ltd).

Progesterone support was started for all patients on the day of ovum pick-up (OPU), Cyclogest® (Alpha Pharm, South Africa, (Pty) Ltd) was administered eight hourly.

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2.1.4 Oocyte Retrieval, Preparation and Fertilization

Female patients underwent transvaginal follicle aspiration using a Cook Oocyte Needle (Cook Medical, Australia) to obtain the oocytes. The resultant embryos were then incubated (37oC, 6% CO2 in air) sequentially with fertilization, cleavage and blastocyst

media (Quinns AdvantageTM_{(Sequential Embryo Media Sage in vitro Fertilization,}

CooperSurgical Company, Trumbull CT, USA).

Oocytes were stripped of their cumulus matrix using hyaluronidase at the concentration of 70 IU (Quinns AdvantageTM Sequential Embryo Media Sage in vitro Fertilization, CooperSurgical Company, Trumbull CT, USA). Metaphase II oocytes were then injected with a spermatozoon from one of the three pre-prepared groups using the most morphologically normal spermatozoon (Figure 6). After 18 hours, fertilization of the oocytes was assessed by the presence of two pronuclei or two polar bodies and the resulting embryos were then placed in sequential media until transfer.

Figure 6: Photo of the ICSI process whereby one spermatozoon is injected into every metaphase II oocyte

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2.1.5 Embryo Culture

On day 2, the embryos were evaluated for cleavage and quality (morphology). On day 3, embryos were evaluated for those patients having a day 3 transfer. Embryos destined for culture to the blastocyst stage, were transferred to blastocyst medium and evaluated for embryo quality. Embryos that cultured to the blastocyst stage were evaluated on day 5 and suitable embryos were transferred. Embryos not transferred (whether day 3 or day 5) were cultured to day 6 and re-evaluated for possible vitrification. Figure 7 shows the stages of embryo culture.

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2.1.6 Transfer

One, two or three, day 3 or 5 embryos were transferred into the uterus with a Cook® K-JETS-SIVF. Transfers were performed using ultrasound guidance of the uterus, thus ensuring that the embryos were deposited approximately 1cm from the fundus. The laboratory personnel then checked the loading catheter as well as the feeding catheters to ensure the embryos had been safely deposited.

2.2 Semen Samples

Teratozoospermic semen samples with normal count and good progressive motility parameters (>30%) according to the World Health Organization (WHO, 2010) criteria were used. After liquefaction a routine semen analysis including evaluation of spermatozoa motility, concentration, vitality and morphology was performed.

2.2.1 Semen Analysis

Spermatozoa concentration was determined with the use of a Bright field Neubauer

hemocytometer and adjusted to a concentration of 20 × 106 motile spermatozoa/ml balanced salt solution (Earle’s buffered saline solution, EBSS or Hams’ F10).

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Motility assessment began after complete liquefaction of the semen to avoid temperature drop

or dehydration of the preparation (Menkveld and Kruger 1996; Menkveld and Coetzee, 1995). The freshly made wet preparation was left to stabilize for approximately one minute. Since spermatozoa motility and velocity are highly dependent on temperature, the assessment was performed at 37ºC, using a heated stage. Examinations were carried out at 20-24ºC, with the temperature in the laboratory standardized, as it could affect the classification of the grades of motility.

1. Using a micro pipette 10l undiluted semen (37C) was placed on a clean microscope slide (37C) and covered by a coverslip (22 x 22 mm).

2. Examination of the “wet preparation” began as soon as the “flow” had stopped (40 x, phase contrast).

3. At least 200 spermatozoa were classified in at least 5 fields.

Forward progression was assessed and quantified as seen below

0 no movement

1 movement - none forward 1+ movement - a few now and then 2 movement - undirected, slow

2+ movement - slowly but directly forward 3- movement - fast but undirected

3 movement - fast and directly forward 3+ movement - very fast and directly forward

4 movement - extremely fast and directly forward (wave-like)

Morphological assessment was done according to the WHO (2010) criteria and is explained

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with a smooth contour. The head should be 4-5µm in length and 2.5-3µm in width leading to a length to width ratio of 1.5:1.75 (Franken and Oehninger, 2012). There should also be a well-defined acrosomal region (40-70% of the head), the mid-piece should be lean and the tail should be straight, uniform and leaner than the mid-piece (explained in section 2.3.1 on page 42).

2.2.2 Semen Preparation for ICSI

Patients undergoing ICSI as method of treatment in the ART arena were randomized and allocated into 3 groups. Each couple was assigned a semen preparation technique according to their randomization and the preparation was as follows:

2.2.2.1 Swim-up or Gradient Preparation

Semen samples that were assigned to this group had either the swim-up or gradient method used for sample preparation. The gradient method was selected if the sample displayed excessive leukocytes in the raw sample. Semen samples were prepared by adding 2ml Sperm Preparation Media (Quinns AdvantageTM Sequential Embryo Media Sage in vitro Fertilization, CooperSurgical Company, Trumbull CT, USA) to the sample which was centrifuged for 10 minutes at 400G using a Heraeus Centrifuge. Once centrifuged, the supernatant was removed and another 2ml of Sperm Preparation Media added repeating the centrifugation process. After the second spin, the supernatant was again removed and 0.7ml of Sperm Preparation Media was loaded above the pellet (without disturbing it). The spermatozoon was allowed to swim from the pellet into the media above for 1 hour. This supernatant was removed and used for ICSI.

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The gradient method of semen preparation was performed using 100% Density Gradient Medium (Quinns AdvantageTM_{Sequential Embryo Media Sage in vitro Fertilization,}

CooperSurgical Company, Trumbull CT, USA) and diluting it to 90%, 70% and 45% respectively using Sperm Preparation Media (Quinns AdvantageTM Sequential Embryo Media Sage in vitro Fertilization, CooperSurgical Company, Trumbull CT, USA). The different densities were then layered on top of each other, starting with the 90% first (at the bottom), then the 70% and lastly 45% (as seen in Figure 8). The semen sample was subsequently loaded on top of the three layers and centrifuged for 20 minutes at 400G using a Heraeus Centrifuge. After centrifugation, the top two layers of density gradient (45% and 70%), as well as the seminal fluid, were removed and discarded. The spermatozoon pellet that remained was mixed with 2ml of spermatozoa preparation solution and centrifuged for 10 minutes at 400G. The supernatant was removed and another 2ml of spermatozoa preparation media was added to the pellet and again centrifuged. The supernatant was again removed and 0.3ml of spermatozoa preparation media was added to the pellet and mixed thoroughly. These spermatozoa were then used for ICSI.

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Figure 8: An illustration of the Swim-up and Gradient Methods of semen preparation Sperm pellet

Gradient Method of Spermatozoa Selection

90% 45%

70%

Prepared Sperm

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2.2.2.2 Cumulus Oophorus Model

COCs were obtained from females undergoing treatment ICSI. After oocyte retrieval, the cumulus oophorus was dissected mechanically using a sterile syringe needle and a glass pipette. The cumulus oophorus from each patient was pooled (individually) in culture medium supplemented with bovine serum albumin (BSA) before experimentation.

A sterile glass capillary with an inner diameter of ±0.7mm was attached onto a 1ml disposal syringe and pre-warmed in an IVF incubator at 37C with humidified air containing 6% CO2 in air. Culture medium supplemented with BSA was aspirated to a length of 3 cm

from the end of the capillary. This was followed by aspiration of cumulus oophorus, thus forming a cumulus oophorus column of a length determined by the experimental design (Figure 9). This end of the capillary was then placed into a 100l droplet of spermatozoa suspension containing 10 × 106 motile spermatozoa/ml overlaid with mineral oil. The experimental apparatus was kept in the IVF chamber for 1 hour. After incubation, the capillary was cut with a diamond tip pen at the interface between the cumulus oophorus column and the medium column. The medium column thus contained the spermatozoa that had passed through the cumulus oophorus (penetrated spermatozoa). These spermatozoa were expelled into a 0.5ml Eppendorf tube. This sample was then used during the ICSI procedure.

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Figure 9: Experimental cumulus oophorus model (Rijsdijk et al. 2007)

2.2.2.3 PICSI

®

_Dish

The PICSI® Dish Spermatozoa Selection Devices (Biocoat Inc. Horsham, PA, USA with distributors MidAtlantic Diagnostic) are commercially available and the

spermatozoa isolation was done as follows: The hyaluronan microdots were rehydrated using Sperm Preparation Media (Quinns AdvantageTM_{Sequential Embryo Media Sage}

in vitro Fertilization, CooperSurgical Company, Trumbull CT, USA) and then covered in tissue culture oil (room temperature). One to 2µl of spermatozoa was added to the media surrounding the first microdot and left for 5 minutes so as to allow the

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spermatozoa to bind to the hyaluronan droplets. Good levels of adherence can be seen in Figure 10, and adhered spermatozoa was selected from the middle of the droplet area as seen by the graphical representation in Figure 11. Once the spermatozoa injection was performed, other spermatozoa were removed from the HA droplet with the same injection needle, and placed on a slide to air-dry for further testing.

Figure 10: The microdot of the PICSI® dish to which the spermatozoa is adhering. This specific patient showed good adherence (magnification of x 440)

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Figure 11: An Illustration of the use of the PICSI® dish

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2.3. Tests Performed on the Raw and Prepared Samples

The following tests were performed on an aliquot of both the raw and prepared samples of each male patient in each of the three groups.

2.3.1. Morphological Evaluation – Diff-Quick Method

Spermatozoa morphology was evaluated using the following guidelines: For a spermatozoon to be considered normal (Menveld et al. 1990), the spermatozoa head, neck, mid-piece and tail must be normal (Figure 12). The head should be oval in shape. Allowing for the slight shrinkage that fixation and staining induce, the length of the head should be 4.0-5.0µm and the width 2.5-3.5µm. The length-to-width ratio should be 1.50:1.75. These ranges are the 95% confidence intervals limits for Papanicolaou-stained spermatozoa heads (Katz et al. 1986). Estimation of the length and width of the spermatozoa were made with an ocular micrometer. There should be a well-defined acrosomal region comprising 40-70% of the head area. The mid-piece should be slender, less than 1µm in width, about one and a half times the length of the head, and attached axially to the head. The tail should be straight, uniform, thinner than the mid-piece, uncoiled and approximately 45µm long (WHO, 1999). This classification scheme requires that all "borderline" forms be considered abnormal (Kruger et al. 1986, Menkveld et al. 1990). Diff-Quick® staining was employed in the study since the author uses the stain as the standard staining method.

The spermatozoa of all patients in each respective group were collected and slides were made and left to air dry. The slides were all stained using the Diff- Quick®_staining

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solutions, which consists of a fixative, staining solution 1 (eosinophillic xanthene) and staining solution 2 (basophilic thiazine). Two hundred spermatozoa were then evaluated for normal forms using the Tygerberg Strict Criteria (Kruger et al. 1986; Menkveld et al. 1990; Coetzee et al. 1998).

The reference limits for the criteria are as follows: Normal ≥ 5%

Abnormal < 5%

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2.3.2. Chromomycin A

3

(CMA

3

)

Chromatin of mature spermatozoa has undergone stabilization when their histones are replaced by protamines and there is formation of molecular disulphide bonds. The nucleoprotein components can be modified during spermatogenesis and this leads to abnormalities in chromatin packaging. The CMA3 staining technique utilizes the detection

of protamine-deficient (or loosely packed) chromatin as well as nicked DNA, to indicate the likelihood of ART success due to possible DNA damage and the failure of the spermatozoon decondensation after ICSI (Bianchi et al. 1996; Lopes et al. 1998; Sakkas et al. 1995) Spermatozoa was collected from all patients and slides were left to air dry. The slides were then fixed using 3:1 ratio of methanol to glacial acetic acid. A 250μl volume of CMA3 –

ethanol (Sigma Chemicals, St Louis, MO USA Cat 2659) was dropped onto the slide and then placed in a dark cupboard for 20 minutes. The slide was then washed in a magnesium chloride McIlvaines buffer solution (0.25mg/mL in McIlvane’s buffer, pH 7.0 containing 10mM MgCl2) and mounted while still wet with Dabco (Aldridge Chemicals Co,

Milwaukee, US Cat No. 29,073-4) and a coverslip. The slides were then left overnight at 4°C and evaluated the following day using a fluorescein isothiocyanate (FITC) filter and an Eplan 100X objective. A total of 200 spermatozoa were randomly evaluated on each slide (Franken et al. 1999).

The spermatozoa are then evaluated as follows 1. No staining (no fluorescence)

2. Fluorescent band at the equatorial region of the spermatozoon head 3. Faint yellow fluorescence of the entire spermatozoon head

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The results are reported as classes 1 and 2 which together represent mature DNA and classes 3 and 4 are indicative of immature DNA as seen in Figure 13.

Figure 13: A photograph of Chromomycin A3 staining illustrating the differences between the good (dull

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2.3.2. Chlortetracycline (CTC) test

The acrosomal status of spermatozoa was determined by CTC staining as previously described (Yao et al. 2000). Spermatozoa was collected from all patients and slides were made. These slides were fixed using a 1:1 ratio of Tris buffer to 12.5% paraformaldehyde solution. The slides were stained with CTC solution and stored overnight at 4°C (750 µM of CTC in 20 mM of Tris buffer supplemented with 130mM of NaCl and 5mM of cysteine) until counting.

The acrosomal status of 200 spermatozoa was evaluated under a fluorescence microscope with a filter set consisting of an excitation filter BP 450-490, a chromatic beam splitter FT510, and a barrier filter LP520. CTC staining patterns of the spermatozoa head were identified according to the method of Perry et al. (1995).

They are: (1) CTC1, a fluorescent band in the postacrosomal region;

(2) CTC2, a bright fluorescent head with a non-fluorescent postacrosomal region;

(3) CTC3, a bright fluorescent head with a non-fluorescent thin band in the postacrosomal region;

(4) CTC4, uniform head fluorescence; and

(5) CTC5, a decrease in or loss of uniform fluorescence over the head.

CTC1, CTC2, and CTC3 were the uncapacitated patterns. CTC4 is the capacitated pattern and CTC5 is the acrosome-reacted pattern. Only CTC5 was counted in this study (Figure 14).

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2.3.3. Acridine Orange (AO)

The DNA quality of the spermatozoa was determined using the acridine orange (AO) staining method as previously described (Claasens et al. 1992; Klun et al. 2002 and Eggert-Kruse et al. 1998). When AO binds to double-stranded DNA it emits a green fluorescence with single-stranded DNA (or RNA) emitting a red, orange or yellow fluorescence (Figure 15). Slides were fixed using Carnoy’s solution overnight (methanol/acetic acid, v/v 3:1) and then left to air dry. Ten millilitres of 1% AO (diluted in distilled water) was mixed with 40ml of 0.1M citric acid and 2.5ml of 0.3M Na27HPO4 and the slides were stained

for 5 minutes with this solution. The slides were then washed in distilled water and sealed using DPX mountant (Sigma) and counted immediately. Two hundred spermatozoa were

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evaluated for each patient and the result expressed as a percentage. The percentage of red, orange or yellow spermatozoa (single-stranded DNA) were recorded as well as the green (double-stranded) fluorescence spermatozoa.

Figure 15 : A photo of the staining characteristics of the acridine orange stain (AO) Green head = diploidy

Orange head = aneuploidy

2.4. The Rationale

In this study we aim to compare the efficacy of the wash and swim-up method, the cumulus oophorus spermatozoa selection model (Hong et al, 2004; Rijsdijk and Franken, 2007) and the PICSI dish in selecting spermatozoa for the ICSI procedure. The primary aim is to determine which spermatozoa selection method delivers the best results in terms of the chromatin packaging quality, DNA integrity, morphology, capacitation and AR. The secondary aim is to determine which method delivers the best results in the ICSI arena in terms in fertilization rate, cleavage rate,

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embryo quality, pregnancy rate, implantation rate and live birth rate. Figure 16 illustrates the study design.

Figure 16: An illustration of the study design

73 ICSI Patients

223 ICSI Patients

Semen Samples

73 ICSI Patients 77 ICSI Patients

WHO analysis & Tygerberg Strict Criteria Randomized Randomized Cumulus Transversal Capillary Model Normal Semen Preparation: Swim-up / Gradient PICSI® Dish CTC CMA3 AO Morphology CTC CMA3 AO Morphology CTC CMA3 AO Morphology Fertilization Rate Pregnancy Rate Implantation Rate Staining _Staining

Compare staining results

Study Design

CTC CMA3 AO Morphology CTC CMA3 AO Morphology CTC CMA3 AO Morphology Staining Staining